Fluorescent pyrazine derivatives and methods of using the same in assessing renal function

ABSTRACT

The present invention relates to pyrazine derivatives such as those represented by Formulas I and II. X 1  to X 4  of Formulas I and II may be characterized as electron withdrawing groups, while Y 1  to Y 4  of Formulas I and II may be characterized as electron donating groups. Pyrazine derivatives of the present invention may be utilized in assessing organ (e.g. kidney) function. In a particular example, an effective amount of a pyrazine derivative that is capable of being renally cleared may be administered into a patient&#39;s body. The pyrazine derivative may be capable of one or both absorbing and emanating spectral energy of at least about 400 nm (e.g. visible and/or infrared light). At least some of the derivative that is in the body may be exposed to spectral energy and, in turn, spectral energy may emanate from the derivative. This emanating spectral energy may be detected and utilized to determine renal function of the patient.

FIELD OF THE INVENTION

The present invention relates to pyrazine derivatives that may becharacterized as hydrophilic, small molecule dyes capable of absorbingand/or emanating spectral energy in the visible and/or near infraredspectrum. In addition, the present invention relates to methods of usingpyrazine derivatives in the monitoring of renal function.

BACKGROUND

Acute renal failure (ARF) is a common ailment in patients admitted togeneral medical-surgical hospitals. Approximately half of the patientswho develop ARF die, and survivors face marked increases in morbidityand prolonged hospitalization [1]. Early diagnosis is generally believedto be critical, because renal failure is often asymptomatic andtypically requires careful tracking of renal function markers in theblood. Dynamic monitoring of renal function of patients is highlydesirable in order to minimize the risk of acute renal failure broughtabout by various clinical, physiological and pathological conditions[2-6]. Such dynamic monitoring is particularly important in the case ofcritically ill or injured patients, because a large percentage of thesepatients tend to face the risk of multiple organ failure (MOF)potentially resulting in death [7, 8]. MOF is a sequential failure ofthe lungs, liver and kidneys and is incited by one or more of acute lunginjury (ALI), adult respiratory distress syndrome (ARDS),hypermetabolism, hypotension, persistent inflammatory focus and sepsissyndrome. The common histological features of hypotension and shockleading to MOF generally include tissue necrosis, vascular congestion,interstitial and cellular edema, hemorrhage and microthrombi. Thesechanges generally affect the lungs, liver, kidneys, intestine, adrenalglands, brain and pancreas in descending order of frequency [9]. Thetransition from early stages of trauma to clinical MOF generallycorresponds with a particular degree of liver and renal failure as wellas a change in mortality risk from about 30% up to about 50% [10].

Traditionally, renal function of a patient has been determined usingcrude measurements of the patient's urine output and plasma creatininelevels [11-13]. These values are frequently misleading because suchvalues are affected by age, state of hydration, renal perfusion, musclemass, dietary intake, and many other clinical and anthropometricvariables. In addition, a single value obtained several hours aftersampling is difficult to correlate with other important physiologicevents such as blood pressure, cardiac output, state of hydration andother specific clinical events (e.g., hemorrhage, bacteremia, ventilatorsettings and others).

With regard to conventional renal monitoring procedures, anapproximation of a patient's glomerular filtration rate (GFR) can bemade via a 24 hour urine collection procedure that (as the namesuggests) typically requires about 24 hours for urine collection,several more hours for analysis, and a meticulous bedside collectiontechnique. Unfortunately, the undesirably late timing and significantduration of this conventional procedure can reduce the likelihood ofeffectively treating the patient and/or saving the kidney(s). As afurther drawback to this type of procedure, repeat data tends to beequally as cumbersome to obtain as the originally acquired data.

Occasionally, changes in serum creatinine of a patient must be adjustedbased on measurement values such as the patient's urinary electrolytesand osmolality as well as derived calculations such as “renal failureindex” and/or “fractional excretion of sodium.” Such adjustments ofserum creatinine undesirably tend to require contemporaneous collectionof additional samples of serum and urine and, after some delay, furthercalculations. Frequently, dosing of medication is adjusted for renalfunction and thus can be equally as inaccurate, equally delayed, and asdifficult to reassess as the measurement values and calculations uponwhich the dosing is based. Finally, clinical decisions in the criticallyill population are often equally as important in their timing as theyare in their accuracy.

Thus, there is a need to develop improved compositions, devices andmethods for measuring renal function (e.g., GFR) using non-ionizingradiation. The availability of a real-time, accurate, repeatable measureof renal excretion rate using exogenous markers under a variety ofcircumstances would represent a substantial improvement over anycurrently available or widely practiced method. Moreover, since such aninvention would depend heavily on the renal elimination of the exogenousmarker(s), the measurement would ideally be absolute and would, thus,preferably require little or no subjective interpretation based on age,muscle mass, blood pressure and the like. Indeed, such an inventionwould enable assessment of renal function under particular circumstancesat particular moments in time.

It is known that hydrophilic, anionic substances are generally capableof being excreted by the kidneys [14]. Renal clearance typically occursvia two pathways: glomerular filtration and tubular secretion. Tubularsecretion may be characterized as an active transport process, andhence, the substances clearing via this pathway typically exhibitspecific properties with respect to size, charge and lipophilicity.

Most of the substances that pass through the kidneys are filteredthrough the glomerulus (a small intertwined group of capillaries in themalpighian body of the kidney). Examples of exogenous substances capableof clearing the kidney via glomerular filtration (hereinafter referredto as “GFR agents”) are shown in FIG. 1 and include creatinine (1),o-iodohippuran (2), and ^(99m)Tc-DTPA (3) [15-17]. Examples of exogenoussubstance that is capable of undergoing renal clearance via tubularsecretion include ^(99m)Tc-MAG3 (4) and other substances known in theart [15, 18, 19]. ^(99m)Tc-MAG3 (4) is also widely used to assess renalfunction though gamma scintigraphy as well as through renal blood flowmeasurement. As one drawback to the substances illustrated in FIG. 1,o-iodohippuran (2), ^(99m)Tc-DTPA (3) and ^(99m)Tc-MAG3 (4) includeradioisotopes to enable the same to be detected. Even if non-radioactiveanalogs (e.g., such as an analog of o-iodohippuran (2)) or othernon-radioactive substances were to be used for renal functionmonitoring, such monitoring would require the use of undesirableultraviolet radiation for excitation of those substances.

Currently, no reliable, continuous, repeatable method for the assessmentof specific renal function using a non-radioactive, exogenous renalagent is commercially available. Among the non-radioactive methods,fluorescence measurement tends to offer the greatest sensitivity. Inprinciple, there are two general approaches for designing fluorescentrenal agents. The first approach would involve enhancing thefluorescence of known renal agents that are intrinsically poor emitters(e.g. lanthanide metal complexes) [21, 22], and the second approachwould involve transforming highly fluorescent dyes (which areintrinsically lipophilic) into hydrophilic, anionic species to forcethem to clear via the kidneys.

Accordingly, it would be quite desirable to transform highly fluorescentdyes into hydrophilic, anionic species. More particularly, it would bequite desirable to identify appropriate, small, fluorescent moleculesand render such molecules hydrophilic. Examples of dyes capable ofabsorbing light in the visible and/or NIR regions are shown in FIG. 2.These dyes are often relatively large in size, contain multiple aromaticrings, and are highly lipophilic compared to the structures shown inFIG. 1. Large lipophilic molecules almost always clear via thehepatobiliary system and do not readily clear via renal pathways. Forexample, FIG. 3 shows that tetrasulfonated cyanine dye (8 of FIG. 2)exhibits a poor rate of clearance from the blood. In attempts tocircumvent this problem, some dyes have been conjugated to polyanioniccarriers [23, 24]. Although these dye-polymer conjugates generallypossess acceptable renal clearance properties, such polymeric compoundshave other drawbacks such as polydispersity, manufacturing and qualitycontrol issues, and the provocation of undesired immune responses thatmay preclude their use as diagnostic and/or therapeutic substances.Accordingly, development of small, hydrophilic dyes is quite desirableto enable enhanced measurement of renal functioning and clearance.

SUMMARY

The present invention generally relates to the transformation offluorescent dyes into hydrophilic and/or anionic species by substitutingboth electron withdrawing and electron donating substituents (i.e., oneor more of each) to the dyes. For example, one aspect of the presentinvention is directed to rigid, small molecules whose size is preferablysimilar to that of creatinine or o-iodohippuran and rendering suchmolecules hydrophilic by incorporating appropriate polar functionalitiessuch as hydroxyl, carboxyl, sulfonate, phosphonate and the like intotheir backbones. Incidentally, the “backbone” of a molecule is a termthat is frequently used in the art to designate a central portion orcore of the molecular structure. For the purpose of this invention, a“small molecule” is an aromatic or a heteroaromatic compound: (1) thatexhibits a molecular weight less than about 500 Daltons; (2) that iscapable of absorbing spectral energy of at least about 400 nm (e.g.,visible and/or near infrared light); and (3) that is capable ofemanating spectral energy of at least about 400 nm (e.g., visible and/ornear infrared light). Further, a “rigid” molecule refers to a moleculethat undergoes little, if any, internal rotational movement. Pyrazinederivatives of the invention may be desirable for renal applicationsbecause they tend to be cleared from the body via the kidneys, maydemonstrate strong absorption and/or emission/fluorescence in thevisible region, and tend to exhibit significant Stokes shifts. Theseproperties allow great flexibility in both tuning the molecule to thedesired wavelength and introducing a wide variety of substituents toimprove clearance properties.

In a first aspect, the present invention is directed to pyrazinederivatives of Formula I (below). With regard to Formula I, X¹ and X²may, at least in some embodiments, be characterized as electronwithdrawing substituents, and each may independently chosen from thegroup consisting of —CN, —CO₂R¹, —CONR²R³, —COR⁴, —NO₂, —SOR⁵, —SO₂R⁶,—SO₂OR⁷ and —PO₃R⁸R⁹. Further, Y¹ and Y² may, at least in someembodiments, be characterized as electron donating substituents and maybe independently chosen from the group consisting of —OR¹⁰, —SR¹¹,—NR¹²R¹³, —N(R¹⁴)COR⁵ and substituents corresponding to Formula A below.Z¹ may be a direct bond, —CR¹⁶R¹⁷—, —O—, —NR¹⁸—, —NCOR¹⁹—, —S—, —SO— or—SO₂—. “m” and “n” may independently be any appropriate integers. Forinstance, in some embodiments, each of “m” and “n” may independently bebetween 1 and 6 (inclusive). As another example, in some embodiments,each of “m” and “n” may independently be between 1 and 3 (inclusive). R¹to R¹⁹ may be any suitable substituents capable of enhancing biologicaland/or physicochemical properties of pyrazine derivatives of Formula I.For example, for renal function assessment, each of the R groups of R¹to R¹⁹ may independently be any one of a hydrogen atom, an anionicfunctional group (e.g., carboxylate, sulfonate, sulfate, phosphonate andphosphate) and a hydrophilic functional group (e.g., hydroxyl, carboxyl,sulfonyl, sulfonato and phosphonato).

A second aspect of the invention is directed to pyrazine derivatives ofFormula II. With regard to Formula II, X³ and X⁴ may, at least in someembodiments, be characterized as electron withdrawing substituents andmay be independently chosen from the group consisting of —CN, —CO₂R²⁰,—CONR²¹R²², —COR²³, —NO₂, —SOR²⁴, —SO₂R²⁵, —SO₂OR²⁶ and —PO₃R²⁷R²⁸. Bycontrast, Y³ and Y⁴ may, at least in some embodiments, be characterizedas electron donating substituents and may be independently chosen fromthe group consisting of —OR²⁹, —SR³⁰, —NR³¹R³², —N(R³²)COR³⁴ andsubstituents corresponding to Formula B below. Z² is preferably a directbond, —CR³⁵R³⁶—, —O—, —NR³⁷—, —NCOR³⁸—, —S—, —SO— or —SO₂—. “p” and “q”may independently be any appropriate integers. For instance, in someembodiments, each of “p” and “q” may independently be between 1 and 6(inclusive). As another example, in some embodiments, each of “p” and“q” may independently be between 1 and 3 (inclusive). R²⁰ to R³⁸ may beany appropriate substituents capable of enhancing biological and/orphysicochemical properties of pyrazine derivatives of Formula II. Forexample, for renal function assessment, each of the R groups of R²⁰ toR³⁸ may independently be any one of a hydrogen atom, an anionicfunctional group (e.g., carboxylate, sulfonate, sulfate, phosphonate andphosphate) and a hydrophilic functional group (e.g., hydroxyl, carboxyl,sulfonyl, sulfonato and phosphonato).

Yet a third aspect of the invention is directed to methods ofdetermining renal function using pyrazine derivatives such as thosedescribed above with regard to Formulas I and II. In these methods, aneffective amount of a pyrazine derivative is administered into the bodyof a patient (e.g., a mammal such as a human or animal subject).Incidentally, an “effective amount” herein generally refers to an amountof pyrazine derivative that is sufficient to enable renal clearance tobe analyzed. The composition is exposed to at least one of visible andnear infrared light. Due to this exposure of the composition to thevisible and/or infrared light, the composition emanates spectral energythat may be detected by appropriate detection equipment. This spectralenergy emanating from the composition may be detected using anappropriate detection mechanism such as an invasive or non-invasiveoptical probe. Herein, “emanating” or the like refers to spectral energythat is emitted and/or fluoresced from a composition of the invention.Renal function can be determined based the spectral energy that isdetected. For example, an initial amount of the amount of compositionpresent in the body of a patient may be determined by amagnitude/intensity of light emanated from the composition that isdetected (e.g., in the bloodstream). As the composition is cleared fromthe body, the magnitude/intensity of detected light generallydiminishes. Accordingly, a rate at which this magnitude of detectedlight diminishes may be correlated to a renal clearance rate of thepatient. This detection may be done periodically or in substantiallyreal time (providing a substantially continuous monitoring of renalfunction). Indeed, methods of the present invention enable renalfunction/clearance to be determined via detecting a change and/or a rateof change of the detected magnitude of spectral energy (indicative of anamount of the composition that has not been cleared) from the portion ofthe composition that remains in the body.

Yet a fourth aspect of the invention is directed to methods forpreparing 2,5-diaminopyrazine-3,6-dicarboxylic acid. In these methods, ahydrolysis mixture including2,4,6,8-tetrahydroxypyrimido(4,5-g)pteridine or a salt thereof isirradiated with microwaves.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Structures of small molecule renal agents.

FIG. 2: Structures of conventional visible and NIR dyes.

FIG. 3: Blood clearance profile of cyanine tetrasulfonate dye (8).

FIG. 4: Block diagram of an assembly for assessing renal function.

FIG. 5: Graph showing renal clearance profile of a normal rat.

FIG. 6: Graph showing renal clearance profile of a bilaterallynephrectomized rat.

FIG. 7: Graph comparing data of FIGS. 5 and 6.

FIGS. 8A & 8B: Projection view of disodium2,5-diamino-3,6-(dicarboxylato)pyrazine crystals prepared as set forthin Example 16. FIG. 8A is a projection view of the molecule with 50%thermal ellipsoids and FIG. 8B is projection view of the molecule with50% thermal ellipsoids and coordination sphere of the Na atoms.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

The present invention discloses renal function monitoring compounds. Anexample of a particular compound of the invention corresponds to FormulaI below. In this exemplary embodiment, X¹ and X² are electronwithdrawing substituents independently chosen from the group consistingof —CN, —CO₂R¹, —CONR²R³, —COR⁴, —NO₂, —SOR⁵, —SO₂R⁶, —SO₂OR⁷ and—PO₃R⁸R⁹. Y¹ and Y² are independently chosen from the group consistingof —OR¹⁰, —SR¹¹, —NR¹²R¹³, —N(R¹⁴)COR¹⁵ and substituents represented byFormula A. Z¹ is selected from the group consisting of a direct bond,—CR¹⁶R¹⁷—, —O—, —NR¹⁸—, —NCOR¹⁹—, —S—, —SO— and —SO₂—. Each of the Rgroups of R¹ to R¹⁹ are independently selected from the group consistingof hydrogen, C3-C6 polyhydroxylated alkyl, —((CH₂)₂—O—(CH₂)₂—O)_(a)R⁴⁰,C1-C10 alkyl, C5-C10 aryl, C5-C10 heteroaryl, —(CH₂)_(a)OH,—(CH₂)_(a)CO₂H, —(CH₂)_(a)SO₃H, —(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)OSO₃H,—(CH₂)_(a)OSO₃ ⁻, —(CH₂)_(a)NHSO₃H, —(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)PO₃H₂,—(CH₂)_(a)PO₃H⁻, —(CH₂)_(a)PO₃ ⁻, (CH₂)_(a)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻ and—(CH₂)_(a)OPO₃. R⁴⁰ is selected from the group consisting of hydrogen,C1-C10 alkyl, C5-C10 aryl, C5-C10 heteroaryl, —(CH₂)_(a)OH,—(CH₂)_(a)CO₂H, —(CH₂)_(a)SO₃H, —(CH₂)SO₃ ⁻, —(CH₂)_(a)OSO₃H,—(CH₂)_(a)OSO₃ ⁻, —(CH₂)_(a)NHSO₃H, —(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)PO₃H₂,—(CH₂)_(a)PO₃H⁻, —(CH₂)_(a)PO₃ ⁻, —(CH₂)_(a)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻ and—(CH₂)_(a)OPO₃. “m” and “n” independently fall within the range of 1 to6 inclusive in some embodiments, and independently fall within the rangeof 1 to 3 inclusive in some embodiments. “a” is an integer from 1 to 10inclusive in some embodiments, and is an integer from 1 to 6 inclusivein some embodiments.

In some embodiments represented by Formula I, each of X¹ and X² are —CN,—CO₂R¹ or —CONR²R³, each of Y¹ and Y² are —NR¹²R¹³ or the substituent ofFormula A, and Z¹ is a direct bond. In such compositions, each of R¹,R², R³, R¹² and R¹³ is not hydrogen, C1-C10 alkyl or C1-C10 aryl, and m,n, N and Z¹ together do not form a 5- or 6-membered ring.

In some embodiments represented by Formula I, X¹ and X² areindependently selected from the group consisting of —CN, —CO₂R¹,—CONR²R³, —SO₂R⁶ and —SO₂OR⁷. Further, Y¹ and Y² are independentlyselected from the group consisting of —NR¹²R¹³, —N(R¹⁴)COR¹⁵ andsubstituents represented by Formula A. Z¹ is selected from the groupconsisting of a direct bond, —CR¹⁶R¹⁷—, —O—, —NR¹⁸, —NCOR¹⁹—, —S—, —SO—and —SO₂—. The R groups of R¹ to R¹⁹ are each independently selectedfrom the group consisting of hydrogen, C3-C6 polyhydroxylated alkyl,—((CH₂)₂—O—(CH₂)₂—O)_(a)—R⁴⁰, C1-C10 alkyl, C5-C10 heteroaryl, C5-C10aryl, —(CH₂)_(a)OH, —(CH₂)_(a)CO₂H, —(CH₂)_(a)SO₃H and —(CH₂)_(a)SO₃ ⁻.Further, “a”, “m” and “n” fall within a range from 1 to 3 inclusive.

In some embodiments represented by Formula I, X¹ and X² areindependently chosen from the group consisting of —CN, —CO₂R¹ and—CONR²R³. Y¹ and Y² are independently selected from the group consistingof —NR¹²R¹³ and substituents represented by Formula A. Z¹ is selectedfrom the group consisting of a direct bond, —CR¹⁶R¹⁷—, —O—, —NR¹⁸,—NCOR¹⁹—, —S—, —SO— and —SO₂—. Each of the R groups of R¹ to R¹⁹ isindependently selected from the group consisting of hydrogen, C3-C6polyhydroxylated alkyl, —((CH₂)₂—O—(CH₂)₂—O)_(a)—R⁴⁰, C1-C10 alkyl,—(CH₂)_(a)OH and —(CH₂)_(a)CO₂H. Further, “a,” “m” and “n” are within arange from 1 to 3 inclusive.

Another example of a particular compound of the invention corresponds toFormula II below. In this exemplary embodiment, X³ and X⁴ are electronwithdrawing substituents independently selected from the groupconsisting of —CN, —CO₂R²⁰, —CONR²¹R²², —COR²³, —NO₂, —SOR²⁴, —SO₂R²⁵,—SO₂OR²⁶ and —PO₃R²⁷R²⁸. Y³ and Y⁴ are electron donating substituentsindependently selected from the group consisting of —OR²⁹, —SR³,—NR³¹R³², —N(R³³)COR³⁴ and substituents represented by Formula B. Z² isselected from the group consisting of a direct bond, —CR³⁵R³⁶—, —O—,—NR³⁷—, —NCOR³⁸—, —S—, —SO—, and —SO₂—. Each of the R groups of R²⁰ toR³⁸ are independently selected from the group consisting of hydrogen,C3-C6 polyhydroxylated alkyl, —((CH₂)₂—O—(CH₂)₂—O)_(b)—R⁴⁰, C1-C10alkyl, C5-C10 aryl, C5-C10 heteroaryl, —(CH₂)_(b)OH, —(CH₂)_(b)CO₂H,—(CH₂)_(b)SO₃H, —(CH₂)_(b)SO₃ ⁻—(CH₂)_(b)OSO₃H, —(CH₂)_(b)OSO₃ ⁻,—(CH₂)_(b)NHSO₃H, —(CH₂)_(b)NHSO₃ ⁻, —(CH₂)_(b)PO₃H₂, —(CH₂)_(b)PO₃H⁻,—(CH₂)_(b)PO₃ ⁻, —(CH₂)_(b)OPO₃H₂, —(CH₂)_(b)OPO₃H⁻ and —(CH₂)_(b)OPO₃.R⁴⁰ is selected from the group consisting of hydrogen, C1-C10 alkyl,C5-C10 aryl, C5-C10 heteroaryl, —(CH₂)_(b)OH, —(CH₂)_(b)CO₂H,—(CH₂)_(b)SO₃H, —(CH₂)_(b)SO₃ ⁻, —(CH₂)_(b)OSO₃H, —(CH₂)_(b)OSO₃ ⁻,—(CH₂)_(b)NHSO₃H, —(CH₂)_(b)NHSO₃ ⁻, —(CH₂)_(b)PO₃H₂, —(CH₂)_(b)PO₃H⁻,—(CH₂)_(b)PO₃ ⁻, —(CH₂)_(b)OPO₃H₂, —(CH₂)_(b)OPO₃H⁻ and —(CH₂)_(b)OPO₃.“p” and “q” independently fall within the range of 1 to 6 inclusive insome embodiments, and independently fall within the range of 1 to 3inclusive in some embodiments. “b” is an integer from 1 to 10 inclusivein some embodiments, and is an integer from 1 to 6 inclusive in someembodiments.

In some embodiments represented by Formula II, X³ and X⁴ areindependently —CN, —CO₂R²⁰ or —CONR²¹R²²; Y³ and Y⁴ are independently—NR³¹R³² or a substituent of Formula B; and Z² is a direct bond. In suchembodiments, each of R²⁰, R²¹, R²², R³¹ and R³² is independently nothydrogen, C3-C6 polyhydroxylated alkyl, —((CH₂)₂—O—(CH₂)₂—O)_(b)—R⁴⁰,C1-C10 alkyl or C1-C10 aryl. Further, p, q, N and Z² together do notform a 5- or 6-membered ring in such embodiments.

In some embodiments represented by Formula II, X³ and X⁴ areindependently selected from the group consisting of —CN, —CO₂R²⁰,—CONR²¹R²², —SO₂R and —SO₂R²⁶. Y³ and Y⁴ are independently selected fromthe group consisting of —NR³¹R³², —N(R³³)COR³⁴ and substituentsrepresented by Formula B. Z² is selected from the group consisting of adirect bond, —CR³⁵R³⁶—, —O—, —NR³⁷—, —NCOR³⁸—, —S—, —SO— and —SO₂—. Eachof the R groups of R²⁰ to R³⁸ are independently selected from the groupconsisting of hydrogen, C3-C6 polyhydroxylated alkyl,—((CH₂)₂—O—(CH₂)₂—O)_(b)—R⁴⁰, C1-C10 alkyl, C5-C10 aryl, C5-C10heteroaryl —(CH₂)_(b)OH, —(CH₂)_(b)CO₂H, —(CH₂)_(b)SO₃H and—(CH₂)_(b)SO₃ ⁻. In these embodiments, “b”, “p” and “q” independentlyrange from 1 to 3 inclusive.

Some embodiments represented by Formula II have X³ and X⁴ beingindependently selected from the group consisting of —CN, —CO₂R²⁰ and—CONR²¹R²². Each of Y³ and Y⁴ may be —NR³³R³⁴ or a substituentrepresented by Formula B. Z² is selected from the group consisting of adirect bond, —CR¹⁶R¹⁷, —O, —NR¹⁸, —NCOR¹⁹, —S, —SO and —SO₂. R²⁰ to R³⁸are independently selected from the group consisting of hydrogen, C3-C6polyhydroxylated alkyl, —((CH₂)₂—O—(CH₂)₂—O)_(b)—R⁴⁰, C1-C10 alkyl,—(CH₂)_(b)OH and —(CH₂)_(a)CO₂H. “b”, “p” and “q” independently rangefrom 1 to 3 inclusive.

By way of example, and not by way of limitation, compounds of Formula Iand Formula II include the following (other exemplary compounds includethose described in Examples 1-16):

Syntheses of pyrazine derivatives, in general, has been previouslystudied [27] and described [25, 26, 28, 29]. Preparation procedures forat least some of the pyrazine derivatives disclosed herein, usingprocedures similar to the cited references, are described herein inExamples 1-8 and 12. Based on the cited references and the disclosureherein, one of ordinary skill in the art will be readily able to preparecompounds of the invention.

In accordance with one aspect of the present invention, compoundscorresponding to Formula I may be derived from2,5-diaminopyrazine-3,6-dicarboxylic acid which, in turn, may be derivedfrom 5-aminouracil. For example, 5-aminouracil may be treated with aferricyanide in the presence of a base to form, as an intermediate,2,4,6,8-tetrahydroxypyrimido(4,5-g)pteridine (or a salt thereof), thepteridine intermediate is heated and hydrolyzed using a base, and thehydrolysate is then acidified to yield2,5-diaminopyrazine-3,6-dicarboxylic as illustrated in Reaction Scheme1.

wherein each Z is independently hydrogen or a monovalent cation. Forexample, each Z may independently be hydrogen or an alkali metal. In oneexemplary embodiment, each Z is hydrogen. In another exemplaryembodiment, each Z is an alkali metal. In yet another exemplaryembodiment, each Z is lithium, sodium or potassium, but they aredifferent (e.g., one is potassium and the other is lithium or sodium).

The series of reactions illustrated in Reaction Scheme 1 are generallycarried out in a suitable solvent. Typically, the reactions will becarried out in an aqueous system.

In one embodiment, each equivalent of 5-aminouracil is treated withabout 3.0 equivalents of ferricyanide, and the concentration of the baseis about 0.5N in the reaction mixture. The ferricyanide used to treat5-aminouracil may be selected from the group consisting of potassiumferricyanide (K₃Fe(CN)₆), lithium ferricyanide (Li₃Fe(CN)₆), sodiumferricyanide (Na₃Fe(CN)₆), sodium potassium ferricyanide, lithium sodiumferricyanide or lithium potassium ferricyanide. Typically, theferricyanide will be potassium ferricyanide. The base used incombination with the ferricyanide is preferably an alkali metalhydroxide, e.g., sodium or potassium hydroxide. See, for example, Tayloret al., JACS, 77: 2243-2248 (1955).

In a preferred embodiment, the hydrolysis mixture is irradiated withmicrowaves to heat the mixture as the2,4,6,8-tetrahydroxypyrimido(4,5-g)pteridine (or salt thereof) ishydrolyzed. At least in some embodiments, the microwaves will have afrequency within the range of about 300 MHz to 30 GHz, and thehydrolysis mixture (preferably an aqueous hydrolysis mixture) is heatedto a temperature within the range of about 120 to about 180° C. for aperiod of about 30 to about 90 minutes. For example, in someembodiments, the hydrolysis mixture will be irradiated with microwavesto heat the hydrolysis mixture to a temperature of about 120 to about140° C. for about 45 to about 75 minutes. In addition to the2,4,6,8-tetrahydroxypyrimido(4,5-g)pteridine (or salt thereof), thehydrolysis mixture of at least some embodiments will typically containat least about 4.7 equivalents of a base, preferably an alkali metalhydroxide (e.g., potassium or sodium hydroxide). The resultinghydrolysate may then be acidified, preferably with a mineral acid suchas hydrochloric acid, sulfuric acid, or phosphoric acid, more preferablyhydrochloric acid, to provide 2,5-diaminopyrazine-3,6-dicarboxylate.

Methods for the conversion of 2,5-diaminopyrazine-3,6-dicarboxylic acidto other compositions falling within Formula I are known to those ofordinary skill. For example, corresponding2,5-diaminopyrazine-3,6-diesters and corresponding2,5-Bis(N,N-dialkylamino) pyrazine-3,6-diesters may be prepared bytreating 2,5-diaminopyrazine-3,6-dicarboxylic acid with the appropriatealkylating agent(s), for example, a mono- or dialkyl halide as describedin Kim et al., Dyes and Pigments, Vol. 39, pages 341-357 (1998).Alternatively, corresponding 2,5-diaminopyrazine-3,6-dithioesters orcorresponding 2,5-Bis(N,N-dialkylamino) pyrazine-3,6-dithioesters may beprepared by treating the 2,5-diaminopyrazine-3,6-dicarboxylic acid witha thiol, or a thiol and the appropriate alkylating agent, respectively,as described in Kim et al., Dyes and Pigments, Vol. 41, pages 183-191(1999).

It is noteworthy that the alkylation of the electron donating aminogroups in cyano- or carboxypyrazines has a profound effect on electronictransition of the pyrazine chromophore in that the dialkylation of theamino group in 2,5-diamino-3,5-dicyanopyrazine produces largebathochromic shift on the order of about 40-60 nm. It is also noteworthythat the pyrrolidino and piperidino derivatives exhibit substantialdifferences in their UV spectra (e.g., the former may tend to exhibit abathochromic shift of about 34 nm).

One protocol for assessing physiological function of renal cellsincludes administering an effective amount of a pyrazine derivative thatis capable of being renally cleared into a body of a patient. Thispyrazine derivative is hydrophilic and capable of absorbing and/oremanating spectral energy of at least about 400 nm. Examples of suchpyrazine derivates are those represented by Formulas I and II above. Anappropriate dosage of the pyrazine derivative that is administered tothe patient is readily determinable by one of ordinary skill in the artand may vary according to such factors as clinical procedurecontemplated, solubility, bioavailability, and toxicity. By way ofexample, an appropriate dosage generally ranges from about 1 nanomolarto about 100 micromolar. The administration of the pyrazine derivativeto the patient may occur in any of a number of appropriate fashionsincluding, but not limited to: (1) intravenous, intraperitoneal, orsubcutaneous injection or infusion; (2) oral administration; (3)transdermal absorption through the skin; and (4) inhalation.

Still referring to the above-mentioned protocol, the pyrazine derivativein the patient's body is exposed to spectral energy of at least about400 nm (preferably, visible and/or near infrared light). This exposureof the pyrazine derivative to spectral energy preferably occurs whilethe pyrazine derivative is in the body (e.g., in the bloodstream). Dueto this exposure of the pyrazine derivative to the spectral energy, thepyrazine derivative emanates spectral energy (e.g., visible and/or nearinfrared light) that may be detected by appropriate detection equipment.The spectral energy emanated from the pyrazine derivative tends toexhibit a wavelength range greater than a wavelength range absorbed bythe pyrazine derivative. For example, if a composition of the inventionabsorbs light of about 700 nm, the composition may emit light of about745 nm.

Detection of the pyrazine derivative (or more particularly, the lightemanating therefrom) may be achieved through optical fluorescence,absorbance, light scattering or other related procedures known in theart. In some embodiments, this detection of the emanated spectral energymay be characterized as a collection of the emanated spectral energy anda generation of electrical signal indicative of the collected spectralenergy. The mechanism(s) utilized to detect the spectral energy from thecomposition that is present in the body may be designed to detect onlyselected wavelengths (or wavelength ranges) and/or may include one ormore appropriate spectral filters. Various catheters, endoscopes, earclips, hand bands, head bands, forehead sensors, surface coils, fingerprobes and the like may be utilized to expose the pyrazine derivativesto light and/or to detect the light emanating therefrom [30]. Thisdetection of spectral energy may be accomplished at one or more timesintermittently or may be substantially continuous.

Renal function of the patient can be determined based on the detectedspectral energy. This can be achieved by using data indicative of thedetected spectral energy and generating an intensity/time profileindicative of a clearance of the pyrazine derivative from the body. Thisprofile may be correlated to a physiological or pathological condition.For example, the patient's clearance profiles and/or clearance rates maybe compared to known clearance profiles and/or rates to assess thepatient's renal function and to diagnose the patient's physiologicalcondition. In the case of analyzing the presence of the pyrazinederivative in bodily fluids, concentration/time curves may be generatedand analyzed (preferably in real time) using an appropriatemicroprocessor to diagnose renal function.

Physiological function can be assessed by any of a number of proceduressuch as any of the following or similar procedures alone or in anycombination: (1) comparing differences in manners in which normal andimpaired cells remove a composition of the invention from thebloodstream; (2) measuring a rate or an accumulation of a composition ofthe invention in the organs or tissues; and (3) obtaining tomographicimages of organs or tissues having a composition of the inventionassociated therewith. For example, blood pool clearance may be measurednon-invasively from convenient surface capillaries such as those foundin an ear lobe or a finger or can be measured invasively using anappropriate instrument such as an endovascular catheter. Accumulation ofa composition of the invention within cells of interest can be assessedin a similar fashion. Incidentally, a “composition” of the inventionrefers to sterile formulations, aqueous formulations, parenteralformulations and any other formulations including one or more of thepyrazine derivatives of the invention. These compositions of theinvention may include pharmaceutically acceptable diluents, carriers,adjuvants, preservatives, excipients, buffers, and the like. The phrase“pharmaceutically acceptable” means those formulations which are, withinthe scope of sound medical judgment, suitable for use in contact withthe tissues of humans and animals without undue toxicity, irritation,allergic response and the like, and are commensurate with a reasonablebenefit/risk ratio.

A modified pulmonary artery catheter may also be utilized to, interalia, make the desired measurements [32] of spectral energy emanatingfrom a composition of the invention. The ability for a pulmonarycatheter to detect spectral energy emanating from a composition of theinvention is a distinct improvement over current pulmonary arterycatheters that measure only intravascular pressures, cardiac output andother derived measures of blood flow. Traditionally, critically illpatients have been managed using only the above-listed parameters, andtheir treatment has tended to be dependent upon intermittent bloodsampling and testing for assessment of renal function. These traditionalparameters provide for discontinuous data and are frequently misleadingin many patient populations.

Modification of a standard pulmonary artery catheter only requiresmaking a fiber optic sensor thereof wavelength-specific. Catheters thatincorporate fiber optic technology for measuring mixed venous oxygensaturation exist currently. In one characterization, it may be said thatthe modified pulmonary artery catheter incorporates awavelength-specific optical sensor into a tip of a standard pulmonaryartery catheter. This wavelength-specific optical sensor can be utilizedto monitor renal function specific elimination of a designed opticallydetectable chemical entity such as the compositions of the presentinvention. Thus, by a method analogous to a dye dilution curve,real-time renal function can be monitored by the disappearance/clearanceof an optically detected compound.

The following examples illustrate specific embodiments of thisinvention. As would be apparent to skilled artisans, various changes andmodifications are possible and are contemplated within the scope of theinvention described.

Example 1 Prophetic Preparation of3,6-dicyano-2,5-[(N,N,N′,N′-tetrakis(carboxymethyl)amino]pyrazine

Step 1.

A stirring mixture of 2,5-diamino-3,6-dicyanopyrazine (10 mmol) andt-butyl bromoacetate (42 mmol) in distilled dimethylacetamide (25 mL) iscooled in ice and subsequently treated with powdered sodium hydroxide(50 mmol). After stirring at ambient temperature for about 2 hours, thereaction mixture is treated water (200 mL) and methylene chloride (100mL). An organic layer of the mixture is washed with copious water, nextdried over sodium sulfate, then filtered, and subsequently the filtrateevaporated in vacuo. The crude product is then purified by flashchromatography to give tetra-t-butyl ester.

Step 2.

The tetraester from Step 1 (10 mmol) is treated with 96% formic acid (10mL) and heated to boiling for about 1 minute and kept at about 40-50° C.for approximately 16 hours. The reaction mixture is poured onto ethercausing formation of a precipitate. This resulting precipitate isseparated from the ether layer by decantation, and then purified bychromatography or recrystallization.

Example 2 Prophetic Preparation of3,6-[(N,N,N′,N′-tetrakis(2-hydroxyethyl)amino]pyrazine-2,5-dicarboxylicacid

Step 1.

The alkylation procedure is identical to the one in Step 1 of Example 1,except that 2-iodoethanol is used instead of t-butylbromoacetate.

Step 2.

The dicyano compound from Step 1 (10 mmol) is dissolved in concentratedsulfuric acid (10 mL) and stirred at ambient temperature for about 3hours. The reaction mixture is carefully diluted with water (100 mL),and the product is collected by filtration and subsequently dried togive the corresponding carboxamide intermediate.

Step 3.

The biscarboxamide derivative from Step 2 (10 mmol) is dissolved inpotassium hydroxide solution (25 mmol in 25 mL of water) and heatedunder reflux for about 3 hours. After cooling, the solution is acidifiedwith 1N HCl (25 mL). The product is collected by filtration, dried, andpurified by recrystallization or chromatography.

Preparation of3,5-[(N,N,N′,N′-tetrakis(2-hydroxyethyl)amino]-pyrazine-2,6-dicarboxylicacid (compound of Formula II) can be accomplished in a similar mannerusing 2,6-diamino-3,5-dicyanopyrazine as the starting material.

Alternatively,3,6-[(N,N,N′,N′-tetrakis(2-hydroxyethyl)amino]pyrazine-2,5-dicarboxylicacid may be prepared by N-alkylating3,6-diaminopyrazine-2,5-dicarboxylic acid (Example 16) with2-iodoethanol as described in Step 1.

Example 3 Prophetic Preparation of3,6-bis(N-azetadino)pyrazine-2,5-dicarboxylic acid

Step 1.

The alkylation procedure is substantially identical to the one in Step 1of Example 1, except that 1,3-dibromopropane is used instead oft-butylbromoacetate.

Step 2.

The hydrolysis procedure is substantially identical to the one in Step 2of Example 2, except that the starting material is3,6-dicyano-2,5-bis(N-azetadino)pyrazine.

Step 3.

The hydrolysis procedure is substantially identical to the one in Step 3of Example 2, except that the starting material is3,6-bis(N-azetadino)-2,5-pyrazinedicarboxamide.

Preparation of 3,5-bis(N-azetadino)pyrazine-2,6-dicarboxylic acid(compound of Formula II) can be accomplished in a similar fashion using2,6-diamino-3,5-dicyanopyrazine as the starting material.

Alternatively, 3,6-bis(N-azetadino)pyrazine-2,5-dicarboxylic acid may beprepared by N-alkylating 3,6-diaminopyrazine-2,5-dicarboxylic acid(Example 16) with 1,3-dibromopropane as described in Step 1.

Example 4 Prophetic Preparation of3,6-bis(N-morpholino)pyrazine-2,5-dicarboxylic acid

Step 1.

The alkylation procedure is identical to the one in Step 1, Example 1except that bis(2-chloroethyl) ether is used instead oft-butylbromoacetate.

Step 2.

The hydrolysis procedure is identical to the one in Step 2, Example 2except that the starting material is3,6-dicyano-2,5-bis(N-morpholino)pyrazine.

Step 3.

The hydrolysis procedure is identical to the one in Step 3, Example 2except that the starting material is3,6-bis(N-morpholino)-2,5-pyrazinedicarboxamide.

Preparation of 3,5-bis(N-morpholino)pyrazine-2,6-dicarboxylic acid(compound belonging to Formula II) can be accomplished in the samemanner using 2,6-diamino-3,5-dicyanopyrazine as the starting material.

Alternatively, 3,6-bis(N-morpholino)pyrazine-2,5-dicarboxylic acid maybe prepared by N-alkylating 3,6-diaminopyrazine-2,5-dicarboxylic acid(Example 16) with (2-chloroethyl) ether as described in Step 1.

Example 5 Prophetic Preparation of3,6-bis(N-piperazino)pyrazine-2,5-dicarboxylic acid

Step 1.

The alkylation procedure is identical to the one in Step 1, Example 1except that bis(2-chloroethyl) amine is used instead oft-butylbromoacetate.

Step 2.

The hydrolysis procedure is identical to the one in Step 2, Example 2except that the starting material is3,6-dicyano-2,5-bis(N-piperazino)pyrazine.

Step 3.

The hydrolysis procedure is identical to the one in Step 3, Example 2except that the starting material is3,6-bis(N-piperazino)-2,5-pyrazinedicarboxamide.

Preparation of 3,5-bis(N-piperazino)pyrazine-2,6-dicarboxylic acid(compound belonging to Formula II) can be accomplished in the samemanner using 2,6-diamino-3,5-dicyanopyazine as the starting material.

Alternatively, 3,6-bis(N-piperazino)pyrazine-2,5-dicarboxylic acid maybe prepared by N-alkylating 3,6-diaminopyrazine-2,5-dicarboxylic acid(Example 16) with bis(2-chloroethyl) amine as described in Step 1.

Example 6 Prophetic Preparation of3,6-bis(N-thiomorpholino)pyrazine-2,5-dicarboxylic acid

Step 1.

A mixture of the tetralcohol product from Step 1, Example 2, (10 mmol),and triethylamine (44 mmol) in anhydrous tetrahydrofuran (50 mL) cooledto 0° C. and treated with methanesulfonyl chloride (42 mmol) added inportion in such a manner that the temperature is maintained at 0 to 15°C. After the addition, the reaction mixture is stirred at ambienttemperature for 16 hours. The reaction mixture is then filtered and thefiltrate taken to dryness under reduced pressure. The residue is thenredissolved in methanol (20 mL) and treated with sodium sulfide (22mmol). The reaction mixture is then heated under reflux for 16 hours andpoured onto water (100 mL) and extracted with ethyl acetate. Thecombined organic layer is washed with copious water, dried over sodiumsulfate, filtered, and the filtrate evaporated in vacuo. The crudeproduct is then purified by flash chromatography to give thebis(thiomorpholino)pyrazine diester.

Step 2.

The hydrolysis procedure is identical to the one in Step 2, Example 2except that the starting material is3,6-dicyano-2,5-bis(N-thiomorpholino)pyrazine.

Step 3.

The hydrolysis procedure is identical to the one in Step 3, Example 2except that the starting material is3,6-bis(N-thiomorpholino)-2,5-pyrazinedicarboxamide.

Preparation of 3,5-bis(N-thiomorpholino)pyrazine-2,6-dicarboxylic acid(compound belonging to Formula II) can be accomplished in the samemanner using 2,6-diamino-3,5-dicyanopyazine as the starting material.

Example 7 Prophetic Preparation of3,6-bis(N-thiomorpholino)pyrazine-2,5-dicarboxylic acid S-oxide

Step 1.

The bis(thiomorpholino)pyrazine derivative from Step 3, Example 6 (5mmol) is dissolved in methanol (20 mL) and treated withm-chloroperoxybenzoic acid (11 mmol) and heated under reflux for 16hours. The reaction mixture poured onto saturated sodium bicarbonate (20mL) and extracted with methylene chloride. The combined organic layer iswashed with brine, dried over sodium sulfate, filtered, and the filtrateevaporated in vacuo. The crude product is purified by chromatography orrecrystallization.

Step 2.

The procedure is identical to Step 2, Example 6 except thatthiomorpholino-S-oxide is used in this experiment.

Preparation of 3,5-bis(N-thiomorpholino)pyrazine-2,6-dicarboxylic acidS-oxide (compound belonging to Formula II) can be accomplished in thesame manner using 2,6-diamino-3,5-dicyanopyazine as the startingmaterial, followed by hydrolysis of the nitrile as outlined in Example1, Step 2 or Example 2, Steps 2 and 3.

Example 8 Prophetic Preparation of2,5-dicyano-3,6-bis(N-thiomorpholino)pyrazine S,S-dioxide

Step 1.

The procedure is identical to Step 1, Example 7 except thatthiomorpholino-S-oxide is used in this experiment.

Step 2.

The procedure is identical to Step 2, Example 6 except thatthiomorpholino-S,S-dioxide is used in this experiment.

Example 9 Prophetic Protocol for Assessing Renal Function

An example of an in vivo renal monitoring assembly 10 is shown in FIG. 4and includes a light source 12 and a data processing. The light source12 generally includes or is interconnected with an appropriate devicefor exposing at least a portion of a patient's body to light therefrom.Examples of appropriate devices that may be interconnected with or be apart of the light source 12 include, but are not limited to, catheters,endoscopes, fiber optics, ear clips, hand bands, head bands, foreheadsensors, surface coils, and finger probes. Indeed, any of a number ofdevices capable of emitting visible and/or near infrared light of thelight source may be employed in the renal monitoring assembly 10.

Still referring to FIG. 4, the data processing system 14 of the renalmonitoring assembly 10 may be any appropriate system capable ofdetecting spectral energy and processing data indicative of the spectralenergy. For instance, the data processing system 14 may include one ormore lenses (e.g., to direct and/or focus spectral energy), one or morefilters (e.g., to filter out undesired wavelengths of spectral energy),a photodiode (e.g., to collect the spectral energy and convert the sameinto electrical signal indicative of the detected spectral energy), anamplifier (e.g., to amplify electrical signal from the photodiode), anda processing unit (e.g., to process the electrical signal from thephotodiode). This data processing system 14 is preferably configured tomanipulate collected spectral data and generate an intensity/timeprofile and/or a concentration/time curve indicative of renal clearanceof a pyrazine composition of the present invention from the patient 20.Indeed, the data processing system 14 may be configured to generateappropriate renal function data by comparing differences in manners inwhich normal and impaired cells remove the pyrazine composition from thebloodstream, to determine a rate or an accumulation of the compositionin organs or tissues of the patient 20, and/or to provide tomographicimages of organs or tissues having the pyrazine composition associatedtherewith.

In one protocol for determining renal function, an effective amount of acomposition including a pyrazine derivative of the invention isadministered to the patient. At least a portion of the body of thepatient 20 is exposed to visible and/or near infrared light from thelight source 12 as indicated by arrow 16. For instance, the light fromthe light source 12 may be delivered via a fiber optic that is affixedto an ear of the patient 20. The patient may be exposed to the lightfrom the light source 12 before or after administration of thecomposition to the patient 20. In some cases, it may be beneficial togenerate a background or baseline reading of light being emitted fromthe body of the patient 20 (due to exposure to the light from the lightsource 12) before administering the composition to the patient 20. Whenthe pyrazine derivative(s) of the composition that are in the body ofthe patient 20 are exposed to the light from the light source 12, thepyrazine derivative(s) emanate light (indicated by arrow 18) that isdetected/collected by the data processing system 14. Initially,administration of the composition to the patient 20 generally enables aninitial spectral signal indicative of the initial content of thepyrazine derivative(s) in the patient 20. The spectral signal then tendsto decay as a function of time as the pyrazine derivative(s) is clearedfrom the patient 20. This decay in the spectral signal as a function oftime is indicative of the patient's renal function. For example, in afirst patient exhibiting healthy/normal renal function, the spectralsignal may decay back to a baseline in a time of T. However, a spectralsignal indicative of a second patient exhibiting deficient renalfunction may decay back to a baseline in a time of T+4 hours. As such,the patient 20 may be exposed to the light from the light source 12 forany amount of time appropriate for providing the desired renal functiondata. Likewise, the data processing system 14 may be allowed tocollect/detect spectral energy for any amount of time appropriate forproviding the desired renal function data.

Example 10 Actual Assessment of Renal Function of Normal Rat

Incident laser light having a wavelength of about 470 nm was deliveredfrom a fiber optic bundle to the ear of an anesthetized Sprague-Dawleyrat. While the light was being directed at the ear, data was beingacquired using a photodector to detect fluorescence coming from withinthe ear. A background reading of fluorescence was obtained prior toadministration of the pyrazine agent. Next, the pyrazine agent (in thiscase, 2 ml of a 0.4 mg/ml solution of3,6-diaminopyrazine-2,5-dicarboxylic acid in PBS) (Example 16) wasadministered into the rat through a bolus injection in the lateral tailvein. As shown in FIG. 5, shortly after the injection, the detectedfluorescence signal rapidly increased to a peak value. The signal thendecayed as a function of time indicating the dye being cleared from thebloodstream (in this case, over a duration of a little over 20 minutes).

The blood clearance time profiles reported herein were assumed to followa two compartment pharmacokinetic model. The fluorescent signal (arisingfrom the dye concentration in the blood) as a function of time wastherefore fit to a double exponential decay. The equation employed tofit the data was:

S=Ae ^(−t/τ) ¹ +Be ^(−t/τ) ² +C  (1)

where S is the fluorescent light intensity signal measured, t is thetime point of the measurement, and e refers to the mathematical constanthaving a numerical value of about 2.71828182846. The decay times τ₁ andτ₂, and the constants A, B, and C are deduced from the fittingprocedure. The non-linear regression analysis package within SigmaPlot®(Systat Software Inc., Richmond, Calif.) was employed to fit data to Eq.(1). In Examples 10 and 11, τ₁ represents the time constant forvascular-extracellular fluid equilibrium, and τ₂ represents the dyeclearance from the blood.

Example 11 Actual Assessment of Renal Function of BilaterallyNephrectomized Rat

An anesthetized Sprague-Dawley rat was bilaterally nephrectomized.Incident laser light having a wavelength of about 470 nm was deliveredfrom a fiber optic bundle to the ear of rat. While the light was beingdirected at the ear, data was being acquired using a photodector todetect fluorescence coming from within the ear. A background reading offluorescence was obtained prior to administration of the pyrazine agent.Next, the pyrazine agent (again, in this case, 2 ml of a 0.4 mg/mlsolution of 3,6-diaminopyrazine-2,5-dicarboxylic acid in PBS) wasadministered into the rat through a bolus injection in the lateral tailvein. As shown in FIG. 6, shortly after the injection, the detectedfluorescence signal rapidly increased to a peak value. However, in thiscase, the pyrazine agent did not clear, indicating that the agent iscapable of being renally cleared. A comparison between the rat thatexhibited normal kidney function (FIG. 5) and the rat that had abilateral nephrectomy (FIG. 6) is shown in FIG. 7. Incidentally,experiments similar to those of Examples 10 and 11 can be utilized todetermine whether or not other proposed agents are capable of beingrenally cleared.

Example 12 Actual Preparation of3,6-dicyano-2,5-[(N,N,N′,N′-tetrakis(carboxymethyl)amino]pyrazine

Step 1.

A stirring mixture of 2,5-diamino-3,6-dicyanopyrazine (1 mmol) andt-butyl bromoacetate (16 mmol) in dimethylacetamide (5 mL) was cooled inan ice-water-bath and subsequently treated with powdered NaOH (6 mmol).The contents were allowed to warm to ambient temperature over 1 h, thenthe reaction mixture was treated with deionized water (50 mL). Thisaqueous mixture was extracted twice with methylene chloride (50 mL). Thecombined organic extracts were dried over sodium sulfate, filtered, andconcentrated in vacuo to afford an oil. This oil was purified by flashchromatography to give the tetra-t-butyl ester.

Step 2.

The tetraester from Step 1 (0.86 mmol) was heated in glacial acetic acid(50 mL) for 24 hours, then was allowed to cool to ambient temperature.The solution was filtered and concentrated in vacuo to afford an oil.The oil was purified by preparative HPLC to afford the title compound.

Example 13 Prophetic Preparation of2,6-dicyano-3,5-[(N,N,N′,N′-tetrakis(hydroxyethyl)amino]pyrazine

To a stirring solution of mixture of tetracyanopyrazine (10 mmol) intetrahydrofuran (25 mL) is treated with dropwise addition ofdiethanolamine (50 mmol) over 30 minutes. After the addition, themixture is stirred at ambient temperature for additional 1 hour. Thecrude product is collected by filtration and purified by chromatographyor recrystallization.

Example 14 Prophetic Preparation of2,6-dicyano-3,5-[(N,N′-bis(hydroxyethyl)amino]pyrazine

The procedure is identical to Example 13 except that aminopropanediol isused in instead of diethanolamine.

Example 15 Prophetic Preparation of2,6-dicyano-3,5-[(N,N′-bis(prolyl)amino]pyrazine

The procedure is identical to Example 13 except that proline is used ininstead of diethanolamine.

Example 16 Actual Synthesis of 3,6-diaminopyrazine-2,5-dicarboxylic acid

Dipotassium 2,4,6,8-tetrahydroxypyrimido(4,5-g)pteridine was prepared bytreating 5-aminouracil with potassium ferricyanide in the presence ofpotassium hydroxide as described in Taylor et al., JACS, 77: 2243-2248(1955).

In each of two Teflon reaction vessels was placed 0.5 g dipotassium2,4,6,8-tetrahydroxypyrimido[4,5g]pteridine and a solution consisting of0.3-0.4 g sodium hydroxide in about 10 mL deionized water. The vesselswere secured in the microwave reactor and allowed to react for one hourat 170° C., generating ca. 100 psi pressure, for one hour. The vesselswere allowed to cool in the microwave to ca. 50° C. and the contentsfiltered to remove a small amount of solid residue. The bright yellowfiltrate was transferred to a 250 mL round-bottom flask equipped with alarge magnetic stir bar. With stirring, the pH was adjusted to ca. 3with concentrated HCl. A large amount of red precipitate formed. A fewmore drops of acid was added and the solid collected by filtration on aglass frit, washed with cold 1×10 mL 1N HCl, 2×30 mL acetonitrile and1×30 mL diethyl ether, suctioned dry and transferred to a vacuum oven,vacuum drying overnight at 45-50° C. Yield 0.48 g (79%). C13 NMR(D₂O/NaOD, external TMS reference) δ 132.35, 147.32, 171.68.

An aliquot of the bright yellow solution was concentrated in vacuoresulting in the formation of two sets of crystals: red needles andyellow blocks. X-Ray crystallography revealed that both crystals aredisodium 2,5-diamino-3,6-(dicarboxylato)pyrazine. The crystal data andstructure refinement for the two sets of crystals are set forth inTables 1R-6R (red crystals) and Tables 1Y-6Y (yellow blocks). Theirstructures are shown in FIG. 8A (projection view of the molecule with50% thermal ellipsoids) and 8B (projection view of the molecule with 50%thermal ellipsoids and coordination sphere of the Na atoms).

Example 17 Prophetic Preparation of 2,5-dicyano3,6-[(N,N′-bis(2,3-dihydroxyhydroxypropyl)amino]-pyrazine

The alkylation procedure is identical to the one in Step 1 of Example 1,except that 3-bromo-1,2-propanediol is used instead oft-butylbromoacetate.

Example 18 Prophetic Preparation of3,6-[(N,N′-bis(2,3-dihydroxypropyl)amino]pyrazine-2,5-dicarboxylic acid

Step 1.

The alkylation procedure is identical to the one in Step 1 of Example 1,except that 3-bromo-1,2-propanediol is used instead oft-butylbromoacetate.

Step 2.

The hydrolysis procedure is identical to the one in Step 2 of Example 2,except that the starting material is the cyano compound in Example 17.

Step 3.

The hydrolysis procedure is identical to the one in Step 3.

Example 19 Prophetic Preparation of 2,5-dicyano3,6-[(N,N′-bis(2,3-dihydroxyhydroxypropyl)amino]-N,N′-dimethylaminopyrazine

The cyano compound (10 mmol) from Example 17 is dissolved indimethylformamide (10 mL) and treated with dimethylsulfate (30 mmol).The mixture is heated at 100° C. for 4 hours and triturated with acetone(100 mL). The crude product is then collected and purified by eithercrystallization or chromatography.

Example 20 Prophetic Preparation of3,6-[(N,N-bis(dimethylamino]pyrazine-2,5-dicarboxylic acid

The title compound is prepared by the hydrolysis of the correspondingdicyano compound by the procedure described in Steps 2 and 3 of Example2.

Example 21 Prophetic Preparation of 2,5-dicyano3,6-[(N,N′-bis(2-sulfonatoethyl)amino]-pyrazine

The alkylation procedure is identical to the one in Step 1 of Example 1,except that taurine (2-aminoethanesulfonate) is used instead oft-butylbromoacetate.

Example 22 Prophetic Preparation of2,5-bis[(N,N′-(2-sulfonato)ethyl]carbamoyl-3,6-[(N,N-bis-(dimethylamino)]pyrazine

A mixture of the diacid in Example 20 (10 mmol), taurine (22 mmol) andthe water-soluble carbodiimide, EDC(ethyldimethylaminopropylcarbodiimide) (25 mmol) in water/DMF (1:1) isstirred at ambient temperature for 16 hours. The solvent is evaporatedin vacuo and the crude product is purified by chromatography.

TABLE 1Y Crystal data and structure refinement for dm16005 (yellow).Identification code m16005/lt/B3401P021-yellow Empirical formulaC3H8N2NaO5 Formula weight 175.10 Temperature 100(2) K Wavelength 0.71073Å Crystal system Monoclinic Space group P2₁/c Unit cell dimensions a =10.5000(10) Å α = 90°. b = 5.2583(5) Å β = 103.207(4)°. c = 13.0181(11)Å γ = 90°. Volume 699.75(11) Å³ Z 4 Density (calculated) 1.662 Mg/m³Absorption coefficient 0.204 mm⁻¹ F (000) 364 Crystal size 0.23 × 0.19 ×0.13 mm³ Theta range for data collection 1.99 to 39.00°. Index ranges−18 ≦ h ≦ 17, −9 ≦ k ≦ 9, −22 ≦ l ≦ 23 Reflections collected 17310Independent reflections 4040 [R(int) = 0.04] Completeness to theta =39.00° 99.4% Absorption correction Semi-empirical from equivalents Max.and min. transmission 0.9739 and 0.9545 Refinement method Full-matrixleast-squares on F² Data/restraints/parameters 4040/0/132Goodness-of-fit on F² 1.045 Final R indices [I > 2sigma(I)] R1 = 0.0365,wR2 = 0.0924 R indices (all data) R1 = 0.0514, wR2 = 0.1005 Largestdiff. peak and hole 0.744 and −0.309 e · Å⁻³

TABLE 2Y Atomic coordinates (×10⁴) and equivalent isotropic displacementparameters (Å² × 10³) for dm16005. U(eq) is defined as one third of thetrace of the orthogonalized U^(ij) tensor. x y z U(eq) Na(1) 5013(1)585(1) 3712(1) 10(1) O(1) 6904(1) 3242(1) 4474(1) 11(1) O(2) 8116(1)5088(1) 3474(1) 13(1) O(3) 6108(1) −1620(1) 2599(1) 11(1) O(4) 3129(1)−2477(1) 3412(1) 14(1) O(5) 3823(1) 2092(1) 4915(1) 11(1) N(1) 8824(1)−4(1) 5294(1) 10(1) N(2) 9494(1) −3201(2) 6512(1) 18(1) C(1) 7933(1)3462(1) 4135(1) 9(1) C(2) 9036(1) 1636(1) 4569(1) 9(1) C(3) 9759(1)−1648(1) 5744(1) 10(1)

TABLE 3Y Bond lengths [Å] and angles [°] for dm16005. Na(1)—O(3)2.3511(6) Na(1)—O(5) 2.3532(6) Na(1)—O(3)#1 2.3533(7) Na(1)—O(5)#22.3815(7) Na(1)—O(1) 2.4457(6) Na(1)—O(4) 2.5110(7) Na(1)—Na(1)#23.4155(7) Na(1)—Na(1)#3 4.1027(5) Na(1)—Na(1)#1 4.1027(5) O(1)—C(1)1.2618(8) O(2)—C(1) 1.2592(9) O(3)—Na(1)#3 2.3534(7) O(3)—H(3A)0.869(15) O(3)—H(3B) 0.823(15) O(4)—H(4A) 0.878(17) O(4)—H(4B) 0.827(17)O(5)—Na(1)#2 2.3814(7) O(5)—H(5A) 0.874(16) O(5)—H(5B) 0.871(14)N(1)—C(2) 1.3339(9) N(1)—C(3) 1.3385(9) N(2)—C(3) 1.3687(10) N(2)—H(2A)0.862(14) N(2)—H(2B) 0.891(14) C(1)—C(2) 1.5105(10) C(2)—C(3)#41.4153(10) C(3)—C(2)#4 1.4152(10) O(3)—Na(1)—O(5) 170.15(2)O(3)—Na(1)—O(3)#1 95.449(17) O(5)—Na(1)—O(3)#1 91.07(2)O(3)—Na(1)—O(5)#2 86.08(2) O(5)—Na(1)—O(5)#2 87.66(2)O(3)#1—Na(1)—O(5)#2 177.57(2) O(3)—Na(1)—O(1) 93.75(2) O(5)—Na(1)—O(1)92.47(2) O(3)#1—Na(1)—O(1) 99.33(2) O(5)#2—Na(1)—O(1) 78.66(2)O(3)—Na(1)—O(4) 93.84(2) O(5)—Na(1)—O(4) 78.47(2) O(3)#1—Na(1)—O(4)92.42(2) O(5)#2—Na(1)—O(4) 89.36(2) O(1)—Na(1)—O(4) 165.33(2)O(3)—Na(1)—Na(1)#2 129.13(2) O(5)—Na(1)—Na(1)#2 44.160(16)O(3)#1—Na(1)—Na(1)#2 135.20(2) O(5)#2—Na(1)—Na(1)#2 43.502(15)O(1)—Na(1)—Na(1)#2 83.835(18) O(4)—Na(1)—Na(1)#2 81.636(18)O(3)—Na(1)—Na(1)#3 29.317(15) O(5)—Na(1)—Na(1)#3 144.77(2)O(3)#1—Na(1)—Na(1)#3 85.888(19) O(5)#2—Na(1)—Na(1)#3 96.338(17)O(1)—Na(1)—Na(1)#3 122.678(18) O(4)—Na(1)—Na(1)#3 66.623(15)Na(1)#2—Na(1)—Na(1)#3 129.770(12) O(3)—Na(1)—Na(1)#1 76.118(19)O(5)—Na(1)—Na(1)#1 112.639(17) O(3)#1—Na(1)—Na(1)#1 29.285(14)O(5)#2—Na(1)—Na(1)#1 150.22(2) O(1)—Na(1)—Na(1)#1 78.905(15)O(4)—Na(1)—Na(1)#1 115.15(2) Na(1)#2—Na(1)—Na(1)#1 150.502(13)Na(1)#3—Na(1)—Na(1)#1 79.709(13) C(1)—O(1)—Na(1) 126.28(5)Na(1)—O(3)—Na(1)#3 121.40(3) Na(1)—O(3)—H(3A) 116.0(10)Na(1)#3—O(3)—H(3A) 102.3(10) Na(1)—O(3)—H(3B) 105.9(10)Na(1)#3—O(3)—H(3B) 105.7(10) H(3A)—O(3)—H(3B) 103.9(13) Na(1)—O(4)—H(4A)98.7(11) Na(1)—O(4)—H(4B) 102.9(11) H(4A)—O(4)—H(4B) 109.9(14)Na(1)—O(5)—Na(1)#2 92.34(2) Na(1)—O(5)—H(5A) 114.6(11)Na(1)#2—O(5)—H(5A) 97.3(10) Na(1)—O(5)—H(5B) 131.9(9) Na(1)#2—O(5)—H(5B)111.0(9) H(5A)—O(5)—H(5B) 103.8(13) C(2)—N(1)—C(3) 120.18(6)C(3)—N(2)—H(2A) 119.5(9) C(3)—N(2)—H(2B) 117.4(9) H(2A)—N(2)—H(2B)115.5(12) O(2)—C(1)—O(1) 125.27(7) O(2)—C(1)—C(2) 117.65(6)O(1)—C(1)—C(2) 117.08(6) N(1)—C(2)—C(3)#4 120.73(6) N(1)—C(2)—C(1)116.00(6) C(3)#4—C(2)—C(1) 123.27(6) N(1)—C(3)—N(2) 116.98(6)N(1)—C(3)—C(2)#4 119.09(6) N(2)—C(3)—C(2)#4 123.90(7) Symmetrytransformations used to generate equivalent atoms: #1−x + 1, y + 1/2,−z + 1/2 #2−x + 1, −y, −z + 1 #3−x + 1, y − 1/2, −z + 1/2 #4−x + 2, −y,−z + 1

TABLE 4Y Anisotropic displacement parameters (Å² × 10³) for dm16005. Theanisotropic displacement factor exponent takes the form: −2π²[h²a*²U¹¹ + . . . + 2 h k a* b* U¹²] U¹¹ U²² U³³ U²³ U¹³ U¹² Na(1)  10(1)11(1) 11(1) 0(1) 3(1) 1(1) O(1)  8(1) 10(1) 14(1) 0(1) 4(1) 1(1) O(2)12(1) 13(1) 15(1) 5(1) 4(1) 3(1) O(3) 12(1) 11(1) 11(1) 1(1) 3(1) 2(1)O(4) 17(1) 14(1) 12(1) 0(1) 5(1) 2(1) O(5) 11(1) 10(1) 14(1) −1(1)  5(1)1(1) N(1)  8(1) 10(1) 12(1) 2(1) 3(1) 2(1) N(2) 12(1) 20(1) 23(1) 13(1) 9(1) 6(1) C(1)  8(1)  9(1) 10(1) −1(1)  1(1) 1(1) C(2)  8(1)  9(1) 10(1)1(1) 2(1) 1(1) C(3)  9(1) 11(1) 12(1) 2(1) 4(1) 1(1)

TABLE 5Y Hydrogen coordinates (×10⁴) and isotropic displacementparameters (Å² × 10³) for dm16005. x y z U(eq) H(3A) 6776(14) −2530(30)2911(12) 29(4) H(3B) 6428(13) −520(30) 2284(11) 29(3) H(4A) 2538(16)−1520(30) 3001(14) 40(4) H(4B) 2966(15) −2590(30) 4003(14) 34(4) H(5A)3053(15) 1390(30) 4852(13) 36(4) H(5B) 3726(12) 3610(30) 5152(11) 25(3)H(2A) 9998(13) −4480(30) 6728(11) 22(3) H(2B) 8657(14) −3410(30)6531(11) 30(3)

TABLE 6Y Torsion angles [°] for dm16005. O(3)—Na(1)—O(1)—C(1) 11.94(6)O(5)—Na(1)—O(1)—C(1) −175.71(6) O(3)#1—Na(1)—O(1)—C(1) −84.22(6)O(5)#2—Na(1)—O(1)—C(1) 97.17(6) O(4)—Na(1)—O(1)—C(1) 132.96(9)Na(1)#2—Na(1)—O(1)—C(1) 140.92(6) Na(1)#3—Na(1)—O(1)—C(1) 6.88(6)Na(1)#1—Na(1)—O(1)—C(1) −63.12(6) O(5)—Na(1)—O(3)—Na(1)#3 59.71(15)O(3)#1—Na(1)—O(3)—Na(1)#3 −71.52(4) O(5)#2—Na(1)—O(3)—Na(1)#3 110.37(3)O(1)—Na(1)—O(3)—Na(1)#3 −171.28(3) O(4)—Na(1)—O(3)—Na(1)#3 21.28(3)Na(1)#2—Na(1)—O(3)—Na(1)#3 103.62(3) Na(1)#1—Na(1)—O(3)—Na(1)#3−93.69(3) O(3)—Na(1)—O(5)—Na(1)#2 50.56(15) O(3)#1—Na(1)—O(5)—Na(1)#2−177.93(2) O(5)#2—Na(1)—O(5)—Na(1)#2 0.0 O(1)—Na(1)—O(5)—Na(1)#2−78.54(2) O(4)—Na(1)—O(5)—Na(1)#2 89.82(2) Na(1)#3—Na(1)—O(5)—Na(1)#297.68(3) Na(1)#1—Na(1)—O(5)—Na(1)#2 −157.54(2) Na(1)—O(1)—C(1)—O(2)90.49(8) Na(1)—O(1)—C(1)—C(2) −90.07(7) C(3)—N(1)—C(2)—C(3)#4 0.69(12)C(3)—N(1)—C(2)—C(1) −178.18(6) O(2)—C(1)—C(2)—N(1) 177.82(6)O(1)—C(1)—C(2)—N(1) −1.66(9) O(2)—C(1)—C(2)—C(3)#4 −1.02(10)O(1)—C(1)—C(2)—C(3)#4 179.50(7) C(2)—N(1)—C(3)—N(2) 177.38(7)C(2)—N(1)—C(3)—C(2)#4 −0.67(12) Symmetry transformations used togenerate equivalent atoms: #1−x + 1, y + 1/2, −z + 1/2 #2−x + 1, −y,−z + 1 #3−x + 1, y − 1/2, −z + 1/2 #4−x + 2, −y, −z + 1

TABLE 1R Crystal data and structure refinement for dm16105.Identification code m16105/lt/B3401P021-red Empirical formulaC₆H₈N₄Na2O₆ Formula weight 278.14 Temperature 100(2) K Wavelength0.71073 Å Crystal system Monoclinic Space group C2/c Unit celldimensions a = 20.549(6) Å α = 90°. b = 3.5198(9) Å β = 100.56(2)°. c =13.289(4) Å γ = 90°. Volume 944.9(5) Å³ Z 4 Density (calculated) 1.955Mg/m³ Absorption coefficient 0.245 mm⁻¹ F (000) 568 Crystal size 0.15 ×0.08 × 0.03 mm³ Theta range for data collection 2.02 to 23.29°. Indexranges −22 ≦ h ≦ 22, −3 ≦ k ≦ 3, −14 ≦ l ≦ 14 Reflections collected 5401Independent reflections 673 [R(int) = 0.11] Completeness to theta =23.29° 99.9% Absorption correction None Max. and min. transmission0.9927 and 0.9641 Refinement method Full-matrix least-squares on F²Data/restraints/parameters 673/1/94 Goodness-of-fit on F² 1.128 Final Rindices [I > 2sigma(I)] R1 = 0.0656, wR2 = 0.1678 R indices (all data)R1 = 0.1011, wR2 = 0.1953 Largest diff. peak and hole 0.553 and −0.459 e· Å⁻³

TABLE 2R Atomic coordinates (×10⁴) and equivalent isotropic displacementparameters (Å² × 10³) for dm16105. U(eq) is defined as one third of thetrace of the orthogonalized U^(ij) tensor. x y z U(eq) Na(1)  0 4107(10)−2500   18(1) Na(2) 2500  2500     0 18(1) O(1) 1044(2) 5915(13)−1625(3)  18(1) O(2) 1697(2) 7546(12) −166(3) 17(1) O(3) 2678(2)1457(16) 1788(3) 23(1) N(1)  −24(2) 8853(15) −999(3) 14(1) N(2)−1135(3)  10283(16)  −1427(4)  17(1) C(1) 1146(3) 7295(18) −736(5) 14(2)C(2)  548(3) 8715(18) −334(4) 14(1) C(3) −579(3) 10076(18)  −695(4)14(1)

TABLE 3R Bond lengths [Å] and angles [°] for dm16105. Na(1)—O(1)2.334(4) Na(1)—O(1)#1 2.334(4) Na(1)—N(1) 2.609(5) Na(1)—N(1)#1 2.609(5)Na(1)—N(1)#2 2.727(5) Na(1)—N(1)#3 2.727(5) Na(1)—Na(1)#3  3.5198(9)Na(1)—Na(1)#4  3.5198(9) Na(2)—O(3)#5 2.365(5) Na(2)—O(3) 2.365(5)Na(2)—O(2)#6 2.383(4) Na(2)—O(2)#3 2.383(4) Na(2)—O(2)#5 2.407(4)Na(2)—O(2) 2.407(4) Na(2)—Na(2)#3  3.5198(9) Na(2)—Na(2)#4  3.5198(9)Na(2)—H(3A) 2.63(7)  O(1)—C(1) 1.259(7) O(2)—C(1) 1.244(7) O(2)—Na(2)#42.383(4) O(3)—H(3A) 0.96(8)  O(3)—H(3B) 0.84(11) N(1)—C(2) 1.335(8)N(1)—C(3) 1.350(8) N(1)—Na(1)#4 2.727(5) N(2)—C(3) 1.359(7) N(2)—H(2A)0.87(4)  N(2)—H(2B) 0.87(4)  C(1)—C(2) 1.512(9) C(2)—C(3)#7 1.422(9)C(3)—C(2)#7 1.422(9) O(1)—Na(1)—O(1)#1 148.4(3)   O(1)—Na(1)—N(1)65.72(16)  O(1)#1—Na(1)—N(1) 93.56(17)  O(1)—Na(1)—N(1)#1 93.56(17) O(1)#1—Na(1)—N(1)#1 65.72(16)  N(1)—Na(1)—N(1)#1 100.4(2)  O(1)—Na(1)—N(1)#2 114.29(16)  O(1)#1—Na(1)—N(1)#2 87.62(15) N(1)—Na(1)—N(1)#2 177.1(2)   N(1)#1—Na(1)—N(1)#2 82.51(13) O(1)—Na(1)—N(1)#3 87.62(15)  O(1)#1—Na(1)—N(1)#3 114.29(16) N(1)—Na(1)—N(1)#3 82.51(13)  N(1)#1—Na(1)—N(1)#3 177.1(2)  N(1)#2—Na(1)—N(1)#3 94.6(2)   O(1)—Na(1)—Na(1)#3 105.83(14) O(1)#1—Na(1)—Na(1)#3 105.82(14)  N(1)—Na(1)—Na(1)#3 129.81(12) N(1)#1—Na(1)—Na(1)#3 129.81(12)  N(1)#2—Na(1)—Na(1)#3 47.30(12) N(1)#3—Na(1)—Na(1)#3 47.30(12)  O(1)—Na(1)—Na(1)#4 74.18(14) O(1)#1—Na(1)—Na(1)#4 74.17(14)  N(1)—Na(1)—Na(1)#4 50.19(12) N(1)#1—Na(1)—Na(1)#4 50.19(12)  N(1)#2—Na(1)—Na(1)#4 132.70(12) N(1)#3—Na(1)—Na(1)#4 132.70(12)  Na(1)#3—Na(1)—Na(1)#4 179.998(1) O(3)#5—Na(2)—O(3) 180.0     O(3)#5—Na(2)—O(2)#6 87.51(15) O(3)—Na(2)—O(2)#6 92.49(15)  O(3)#5—Na(2)—O(2)#3 92.49(15) O(3)—Na(2)—O(2)#3 87.51(15)  O(2)#6—Na(2)—O(2)#3 180.0    O(3)#5—Na(2)—O(2)#5 100.60(16)  O(3)—Na(2)—O(2)#5 79.40(16) O(2)#6—Na(2)—O(2)#5 94.58(14)  O(2)#3—Na(2)—O(2)#5 85.42(14) O(3)#5—Na(2)—O(2) 79.40(16)  O(3)—Na(2)—O(2) 100.60(16) O(2)#6—Na(2)—O(2) 85.42(14)  O(2)#3—Na(2)—O(2) 94.58(14) O(2)#5—Na(2)—O(2) 180.0     O(3)#5—Na(2)—Na(2)#3 98.93(14) O(3)—Na(2)—Na(2)#3 81.07(14)  O(2)#6—Na(2)—Na(2)#3 137.03(10) O(2)#3—Na(2)—Na(2)#3 42.97(10)  O(2)#5—Na(2)—Na(2)#3 42.45(10) O(2)—Na(2)—Na(2)#3 137.55(10)  O(3)#5—Na(2)—Na(2)#4 81.07(14) O(3)—Na(2)—Na(2)#4 98.93(14)  O(2)#6—Na(2)—Na(2)#4 42.97(10) O(2)#3—Na(2)—Na(2)#4 137.02(10)  O(2)#5—Na(2)—Na(2)#4 137.56(10) O(2)—Na(2)—Na(2)#4 42.45(10)  Na(2)#3—Na(2)—Na(2)#4 180.0    O(3)#5—Na(2)—H(3A) 158.6(17)   O(3)—Na(2)—H(3A) 21.4(17) O(2)#6—Na(2)—H(3A) 79.2(18)  O(2)#3—Na(2)—H(3A) 100.8(18)  O(2)#5—Na(2)—H(3A) 64.3(18)  O(2)—Na(2)—H(3A) 115.7(18)  Na(2)#3—Na(2)—H(3A) 80.3(18)  Na(2)#4—Na(2)—H(3A) 99.7(18) C(1)—O(1)—Na(1) 123.5(4)   C(1)—O(2)—Na(2)#4 130.2(4)   C(1)—O(2)—Na(2)122.4(4)   Na(2)#4—O(2)—Na(2) 94.58(14)  Na(2)—O(3)—H(3A) 95(4)   Na(2)—O(3)—H(3B) 125(7)     H(3A)—O(3)—H(3B) 99(8)    C(2)—N(1)—C(3)120.2(5)   C(2)—N(1)—Na(1) 110.2(4)   C(3)—N(1)—Na(1) 124.6(4)  C(2)—N(1)—Na(1)#4 112.2(4)   C(3)—N(1)—Na(1)#4 97.6(4)  Na(1)—N(1)—Na(1)#4 82.51(13)  C(3)—N(2)—H(2A) 114(4)     C(3)—N(2)—H(2B)118(4)     H(2A)—N(2)—H(2B) 123(6)     O(2)—C(1)—O(1) 125.1(6)  O(2)—C(1)—C(2) 118.0(5)   O(1)—C(1)—C(2) 116.9(5)   N(1)—C(2)—C(3)#7120.4(6)   N(1)—C(2)—C(1) 116.9(5)   C(3)#7—C(2)—C(1) 122.8(5)  N(1)—C(3)—N(2) 116.7(5)   N(1)—C(3)—C(2)#7 119.4(5)   N(2)—C(3)—C(2)#7123.8(6)   Symmetry transformations used to generate equivalent atoms:#1−x, y, −z − 1/2 #2−x, y − 1, −z − 1/2 #3x, y − 1, z #4x, y + 1, z#5−x + 1/2, −y + 1/2, −z #6−x + 1/2, −y + 3/2, −z #7−x, −y + 2, −z

TABLE 4R Anisotropic displacement parameters (Å² × 10³) for dm16105. Theanisotropic displacement factor exponent takes the form: −2π²[h²a*²U¹¹ + . . . + 2 h k a* b* U¹²] U¹¹ U²² U³³ U²³ U¹³ U¹² Na(1)  23(2)12(2) 19(2) 0 3(1) 0 Na(2)  19(2) 12(2) 24(2) 2(2) 5(2) −1(2) O(1) 21(2)16(3) 17(2) −3(2)  3(2)  1(2) O(2) 20(3)  9(3) 22(2) 0(2) 2(2)  2(2)O(3) 20(3) 25(3) 25(3) −1(2)  7(2) −2(2) N(1) 17(3)  2(3) 22(3) 0(2)4(2) −1(2) N(2) 20(3) 13(4) 19(3) −4(3)  4(3)  3(3) C(1) 16(4)  2(4)22(4) 5(3) 3(3) −2(3) C(2) 19(3)  3(3) 20(2) 5(2) 1(2) −1(2) C(3) 19(3) 3(3) 20(2) 5(2) 1(2) −1(2)

TABLE 5R Hydrogen coordinates (×10⁴) and isotropic displacementparameters (Å² × 10³) for dm16105. x y z U(eq) H(3A)  3150(40) 1200(200) 1860(50) 40(20) H(3B)  2660(50) 3100(300)  2230(70) 80(40) H(2A)−1120(30) 8900(170) −1960(40) 14(17) H(2B) −1510(20) 10860(180) −1240(40) 10(16)

TABLE 6R Torsion angles [°] for dm16105. O(1)#1—Na(1)—O(1)—C(1)  72.9(5)N(1)—Na(1)—O(1)—C(1)  20.0(5) N(1)#1—Na(1)—O(1)—C(1) 119.8(5)N(1)#2—Na(1)—O(1)—C(1) −156.9(5)  N(1)#3—Na(1)—O(1)—C(1) −62.9(5)Na(1)#3—Na(1)—O(1)—C(1) −107.1(5)  Na(1)#4—Na(1)—O(1)—C(1)  72.9(5)O(3)#5—Na(2)—O(2)—C(1)  56.5(4) O(3)—Na(2)—O(2)—C(1) −123.5(4) O(2)#6—Na(2)—O(2)—C(1) 144.8(5) O(2)#3—Na(2)—O(2)—C(1) −35.2(5)O(2)#5—Na(2)—O(2)—C(1)  8(7) Na(2)#3—Na(2)—O(2)—C(1) −35.2(5)Na(2)#4—Na(2)—O(2)—C(1) 144.8(5) O(3)#5—Na(2)—O(2)—Na(2)#4  −88.32(16)O(3)—Na(2)—O(2)—Na(2)#4   91.68(16) O(2)#6—Na(2)—O(2)—Na(2)#4 0.0 O(2)#3—Na(2)—O(2)—Na(2)#4 180.0   O(2)#5—Na(2)—O(2)—Na(2)#4 −137(6)  Na(2)#3—Na(2)—O(2)—Na(2)#4 180.0   O(1)—Na(1)—N(1)—C(2) −21.7(4)O(1)#1—Na(1)—N(1)—C(2) −176.9(4)  N(1)#1—Na(1)—N(1)—C(2) −110.9(4) N(1)#2—Na(1)—N(1)—C(2)  69.1(4) N(1)#3—Na(1)—N(1)—C(2)  69.1(4)Na(1)#3—Na(1)—N(1)—C(2)  69.1(4) Na(1)#4—Na(1)—N(1)—C(2) −110.9(4) O(1)—Na(1)—N(1)—C(3) −176.7(5)  O(1)#1—Na(1)—N(1)—C(3)  28.1(5)N(1)#1—Na(1)—N(1)—C(3)  94.1(5) N(1)#2—Na(1)—N(1)—C(3) −85.9(5)N(1)#3—Na(1)—N(1)—C(3) −85.9(5) Na(1)#3—Na(1)—N(1)—C(3) −85.9(5)Na(1)#4—Na(1)—N(1)—C(3)  94.1(5) O(1)—Na(1)—N(1)—Na(1)#4   89.24(16)O(1)#1—Na(1)—N(1)—Na(1)#4  −65.95(15) N(1)#1—Na(1)—N(1)—Na(1)#4  0.002(1) N(1)#2—Na(1)—N(1)—Na(1)#4   179.998(11)N(1)#3—Na(1)—N(1)—Na(1)#4 180.0   Na(1)#3—Na(1)—N(1)—Na(1)#4 180.0  Na(2)#4—O(2)—C(1)—O(1)  89.2(7) Na(2)—O(2)—C(1)—O(1) −42.1(8)Na(2)#4—O(2)—C(1)—C(2) −91.4(6) Na(2)—O(2)—C(1)—C(2) 137.3(5)Na(1)—O(1)—C(1)—O(2) 164.0(5) Na(1)—O(1)—C(1)—C(2) −15.3(8)C(3)—N(1)—C(2)—C(3)#7  −1.2(10) Na(1)—N(1)—C(2)—C(3)#7 −157.5(5) Na(1)#4—N(1)—C(2)—C(3)#7 112.5(5) C(3)—N(1)—C(2)—C(1) 179.8(5)Na(1)—N(1)—C(2)—C(1)  23.5(7) Na(1)#4—N(1)—C(2)—C(1) −66.5(6)O(2)—C(1)—C(2)—N(1) 172.0(5) O(1)—C(1)—C(2)—N(1)  −8.6(9)O(2)—C(1)—C(2)—C(3)#7  −7.0(9) O(1)—C(1)—C(2)—C(3)#7 172.4(6)C(2)—N(1)—C(3)—N(2) 177.4(5) Na(1)—N(1)—C(3)—N(2) −30.0(8)Na(1)#4—N(1)—C(3)—N(2)  56.1(6) C(2)—N(1)—C(3)—C(2)#7   1.2(10)Na(1)—N(1)—C(3)—C(2)#7 153.9(4) Na(1)#4—N(1)—C(3)—C(2)#7 −120.0(5) Symmetry transformations used to generate equivalent atoms: #1−x, y, −z− 1/2 #2−x, y − 1, −z − 1/2 #3x, y − 1, z #4x, y + 1, z #5−x + 1/2, −y +1/2, −z #6−x + 1/2, −y + 3/2, −z #7−x, −y + 2, −z

Various publications are referenced throughout this disclosure by Arabicnumerals in brackets. A full citation corresponding to each referencenumber is listed below. The disclosures of these publications are hereinincorporated by reference in their entireties.

REFERENCES

-   1. Nally, J. V. Acute renal failure in hospitalized patients.    Cleveland Clinic Journal of Medicine 2002, 69(7), 569-574.-   2. C. A. Rabito, L. S. T. Fang, and A. C. Waltman. Renal function in    patients at risk with contrast material-induced acute renal failure:    Noninvasive real-time monitoring. Radiology 1993, 186, 851-854.-   3. N. L. Tilney, and J. M. Lazarus. Acute renal failure in surgical    patients: Causes, clinical patterns, and care. Surgical Clinics of    North America 1983, 63, 357-377.-   4. B. E. VanZee, W. E. Hoy, and J. R. Jaenike. Renal injury    associated with intravenous pyelography in non-diabetic and diabetic    patients. Annals of Internal Medicine 1978, 89, 51-54.-   5. S. Lundqvist, G. Edbom, S. Groth, U. Stendahl, and S.-O. Hietala.    Iohexol clearance for renal function measurement in gynecologic    cancer patients. Acta Radiologica 1996, 37, 582-586.-   6. P. Guesry, L. Kaufman, S. Orloff, J. A. Nelson, S. Swann, and M.    Holliday. Measurement of glomerular filtration rate by fluorescent    excitation of non-radioactive meglumine iothalamate. Clinical    Nephrology 1975, 3, 134-138).-   7. C. C. Baker et al. Epidemiology of Trauma Deaths. American    Journal of Surgery 1980, 144-150.-   8. R. G. Lobenhoffer et al. Treatment Results of Patients with    Multiple Trauma: An Analysis of 3406 Cases Treated Between 1972 and    1991 at a German Level I Trauma Center. Journal of Trauma 1995, 38,    70-77.-   9. J. Coalson, Pathology of Sepsis, Septic Shock, and Multiple Organ    Failure. In New Horizons: Multiple Organ Failure, D. J. Bihari    and F. B. Cerra, (Eds). Society of Critical Care Medicine,    Fullerton, Calif., 1986, pp. 27-59.-   10. F. B. Cerra, Multiple Organ Failure Syndrome. In New Horizons:    Multiple Organ Failure, D. J Bihari and F. B. Cerra, (Eds). Society    of Critical Care Medicine, Fullerton, Calif., 1989, pp. 1-24.-   11. R. Muller-Suur, and C. Muller-Suur. Glomerular filtration and    tubular secretion of MAG₃ in rat kidney. Journal of Nuclear Medicine    1989, 30, 1986-1991).-   12. P. D. Dollan, E. L. Alpen, and G. B. Theil. A clinical appraisal    of the plasma concentration and endogenous clearance of creatinine.    American Journal of Medicine 1962, 32, 65-79.-   13. J. B. Henry (Ed). Clinical Diagnosis and Management by    Laboratory Methods, 17th Edition, W.B. Saunders, Philadelphia, Pa.,    1984.-   14. F. Roch-Ramel, K. Besseghir, and H. Murer. Renal excretion and    tubular transport of organic anions and cations. In Handbook of    Physiology, Section 8, Neurological Physiology, Vol. II, E. E.    Windhager, Editor, pp. 2189-2262. Oxford University Press: New York,    1992-   15. D. L. Nosco and J. A. Beaty-Nosco. Chemistry of technetium    radiopharmaceuticals 1: Chemistry behind the development of    technetium-99m compounds to determine kidney function. Coordination    Chemistry Reviews 1999, 184, 91-123.-   16. P. L. Choyke, H. A. Austin, and J. A. Frank. Hydrated clearance    of gadolinium-DTPA as a measurement of glomerular filtration rate.    Kidney International 1992, 41, 1595-1598.-   17. N. Lewis, R. Kerr, and C. Van Buren. Comparative evaluation of    urographic contrast media, inulin, and ^(99m)Tc-DTPA clearance    methods for determination of glomerular filtration rate in clinical    transplantation. Transplantation 1989, 48, 790-796).-   18. W. N. Tauxe. Tubular Function. In Nuclear Medicine in Clinical    Urology and Nephrology, W. N. Tauxe and E. V. Dubovsky, Editors, pp.    77-105, Appleton Century Crofts: East Norwalk, 1985.-   19. A. R. Fritzberg et al. Mercaptoacetylglycylglycyglycine. Journal    of Nuclear Medicine 1986, 27, 111-120.-   20. G. Ekanoyan and N. W. Levin. In Clinical Practice Guidelines for    Chronic Kidney Disease: Evaluation, Classification, and    Stratification (K/DOQI). National Kidney Foundation: Washington,    D.C. 2002, pp. 1-22.-   21. Ozaki, H. et al. Sensitization of europium(III) luminescence by    DTPA derivatives. Chemistry Letters 2000, 312-313.-   22. Rabito, C. Fluorescent agents for real-time measurement of organ    function. U.S. patent 2002; U.S. Pat. No. 6,440,389.-   23. R. Rajagopalan, R. et al. Polyionic fluorescent bioconjugates as    composition agents for continuous monitoring of renal function. In    Molecular Imaging: Reporters, Dyes, Markers, and Instrumentation, A.    Priezzhev, T. Asakura, and J. D. Briers, Editors, Proceedings of    SPIE, 2000, 3924.-   24. Dorshow, R. B. et al. Noninvasive renal function assessment by    fluorescence detection. In Biomedical Optical Spectroscopy and    Diagnostics, Trends in Optics and Photonics Series 22, E. M    Sevick-Muraca, J. A. Izatt, and M. N. Ediger, Editors, pp. 54-56,    Optical Society of America, Washington D.C., 1998.-   25. Shirai, K. et al Synthesis and fluorescent properties of    2,5-diamino-3,6-dicyanopyrazine dyes. Dyes and Pigments 1998, 39(1),    49-68.-   26. Kim, J. H. et al. Self-assembling of aminopyrazine fluorescent    dyes and their solid state spectra. Dyes and Pigments 1998, 39(4),    341-357.-   27. Barlin, G. B. The pyrazines. In The Chemistry of Heterocyclic    Compounds. A. Weissberger and E. C. Taylor, Eds. John Wiley & Sons,    New York: 1982.-   28. Donald, D. S. Synthesis of 3,5-diaminopyrazinoic acid from    3,5-diamino-2,6-dicyanopyrazine and intermediates. U.S. patent 1976;    U.S. Pat. No. 3,948,895.-   29. Donald, D. S. Diaminosubstituted dicyanopyrzines and process.    U.S. patent 1974; U.S. Pat. No. 3,814,757.-   30. Muller et al. Eds, Medical Optical Tomography, SPIE Volume IS11,    1993.-   31. R. B. Dorshow et al. Non-Invasive Fluorescence Detection of    Hepatic and Renal Function, Bull. Am. Phys. Soc. 1997, 42, 681.-   32. R. B. Dorshow et al. Monitoring Physiological Function by    Detection of Exogenous Fluorescent Contrast Agents. In Optical    Diagnostics of Biological Fluids IV, A. Priezzhev and T. Asakura,    Editors, Proceedings of SPIE 1999, 3599, 2-8).

1.-44. (canceled)
 45. A compound of Formula I wherein:

each of X¹ and X² is independently selected from the group consisting of—CN, —CO₂R¹, —CONR²R³, —COR⁴, —SOR⁵, —SO₂R⁶, —SO₂OR⁷ and —PO₃R⁸R⁹; eachof Y¹ and Y² is independently selected from the group consisting of—OR¹⁰, —SR¹¹, —NR¹²R¹³, —N(R¹⁴)COR¹⁵ and

Z¹ is selected from the group consisting of a direct bond, —CR¹⁶R¹⁷—,—O—, —NR¹⁸—, —NCOR¹⁹—, —S—, —SO— and —SO₂—; each of R¹ to R⁹, R¹¹, andR¹⁶ to R¹⁹ is independently selected from the group consisting ofhydrogen, C3-C6 polyhydroxylated alkyl, —((CH₂)₂—O—(CH₂)₂—O)_(a)—R⁴⁰,C1-C10 alkyl, C5-C10 aryl, C5-C10 heteroaryl, —(CH₂)_(a)OH,—(CH₂)_(a)CO₂H, —(CH₂)_(a)SO₃H, —(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)OSO₃H,—(CH₂)_(a)OSO₃ ⁻, —(CH₂)_(a)NHSO₃H, —(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)PO₃H₂,—(CH₂)_(a)PO₃H⁻, —(CH₂)_(a)PO₃ ⁼, —(CH₂)_(a)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻ and—(CH₂)_(a)OPO₃; each of R¹² to R¹⁵ is independently selected from C5-C10heteroaryl; R¹⁰ is selected from the group consisting of C3-C6polyhydroxylated alkyl, —((CH₂)₂—O—(CH₂)₂—O)_(a)—R⁴⁰, C1-C10 alkyl,C5-C10 aryl, C5-C10 heteroaryl, —(CH₂)_(a)OH, —(CH₂)_(a)CO₂H,—(CH₂)_(a)SO₃H, —(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)OSO₃H, —(CH₂)_(a)OSO₃ ⁻,—(CH₂)_(a)NHSO₃H, —(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)PO₃H₂, —(CH₂)_(a)PO₃H⁻,—(CH₂)_(a)PO₃ ⁼, —(CH₂)_(a)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻ and —(CH₂)_(a)OPO₃;R⁴⁰ is selected from the group consisting of hydrogen, C1-C10 alkyl,C5-C10 aryl, C5-C10 heteroaryl, —(CH₂)_(a)OH, —(CH₂)_(a)CO₂H,—(CH₂)_(a)SO₃H, —(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)OSO₃H, —(CH₂)_(a)OSO₃ ⁻,—(CH₂)_(a)NHSO₃H, —(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)PO₃H₂, —(CH₂)_(a)PO₃H⁻,—(CH₂)_(a)PO₃ ⁼, —(CH₂)_(a)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻ and —(CH₂)_(a)OPO₃;and each of a, m and n range from 1 to 6; with the proviso that if: eachof X¹ and X² is independently —CN, —CO₂R¹ or —CONR²R³; each of Y¹ and Y²is independently —NR¹²R¹³ or

and Z¹ is a direct bond, then: each of R¹, R², R³, R¹² and R¹³ isindependently not hydrogen, C1-C10 alkyl or C5-C10 aryl; and the sum ofm and n is not 4 or
 5. 46. The compound of claim 45 wherein: each of X¹and X² is independently selected from the group consisting of —CN,—CO₂R¹ and —CONR²R³; each of Y¹ and Y² is independently —NR¹²R¹³ or

each of R¹, R², and R³ is independently selected from the groupconsisting of hydrogen, C3-C6 polyhydroxylated alkyl,—((CH₂)₂—O—(CH₂)₂—O)_(a)—R⁴⁰, C5-C10 heteroaryl —(CH₂)_(a)OH,—(CH₂)_(a)CO₂H, —(CH₂)_(a)SO₃H and —(CH₂)_(a)SO₃ ⁻; each of R¹⁶ to R¹⁹is independently selected from the group consisting of hydrogen, C3-C6polyhydroxylated alkyl, —((CH₂)₂—O—(CH₂)₂—O)_(a)—R⁴⁰, C1-C10 alkyl,C5-C10 aryl, C5-C10 heteroaryl, —(CH₂)_(a)OH, —(CH₂)_(a)CO₂H,—(CH₂)_(a)SO₃H and —(CH₂)_(a)SO₃ ⁻; m is 1 or 2; and n is
 1. 47. Thecompound of claim 46 wherein: each of X¹ and X² is —CN; each of Y¹ andY² is independently selected from the group consisting of —NR¹²R¹³ and

Z¹ is selected from the group consisting of a direct bond, —O—, —NR¹⁸—,—NCOR¹⁹—, —S—, —SO— and —SO₂—; each of R¹⁸ and R¹⁹ is independentlyselected from the group consisting of hydrogen, C3-C6 polyhydroxylatedalkyl, —((CH₂)₂—O—(CH₂)₂—O)_(a)—R⁴⁰, C1-C10 alkyl, —(CH₂)_(a)OH,—(CH₂)_(a)CO₂H, —(CH₂)_(a)SO₃H and —(CH₂)_(a)SO₃ ⁻; m is 1 or 2; and nis
 1. 48. The compound of claim 47 wherein: each of Y¹ and Y² is—NR¹²R¹³.
 49. The compound of claim 47 wherein: each of Y¹ and Y² is


50. The compound of claim 46 wherein: each of X¹ and X² is —CO₂R¹; eachof Y¹ and Y² is independently selected from the group consisting of—NR¹²R¹³ and

Z¹ is selected from the group consisting of a direct bond, —O—, —NR¹⁸—,—NCOR¹⁹—, —S—, —SO— and —SO₂—; R¹ is hydrogen; and each of R¹⁸ and R¹⁹is independently selected from the group consisting of hydrogen, C3-C6polyhydroxylated alkyl, —((CH₂)₂—O—(CH₂)₂—O)_(a)—R⁴⁰, C1-C10 alkyl,—(CH₂)_(a)OH, —(CH₂)_(a)CO₂H, —(CH₂)_(a)SO₃H and —(CH₂)_(a)SO₃ ⁻. 51.The compound of claim 50 wherein: each of Y¹ and Y² is —NR¹²R¹³.
 52. Thecompound of claim 50 wherein: each of Y¹ and Y² is